IL-1, formerly known in the literature as "lymphocyte activating factor" or "LAF," is a hormone secreted by macrophages while undergoing on immune response. This protein factor regulates a wide range of immunological and non-immunological responses. For instance, it is considered that IL-1 mediates activities referred to as endogenous or leukocytic pyrogen, B-cell activating factor (BAF), epidermal cell thymocyte activating factor (ETAF), leukocyte endogenous mediator (LEM), bone resorption factor active in rheumatoid arthritis, and a variety of other activities.
Although researchers have identified many of the biological properties of IL-1, the chemical nature of this hormone is not well understood. To date, this has been hampered, at least in part, by the unavailability of sufficient quantities of IL-1 in purified form to carry out necessary investigations.
Attempts have been made in the past to purify and partially characterize IL-1 derived from both human and murine sources. For instance, Mizel, 122 J. Immunol. 2167-2172 (1979), reported the production of murine IL-1 from the macrophage cell line, P388D.sub.1, cultured in a supplemented growth medium together with phorbol myristic acetate as an activating agent. The IL-1 from the culture fluid was subjected to ammonium sulfate precipitation, diethyl amino ethyl ("DEAE") cellulose column chromatography, ultrafiltration and Sephacryl S200 column chromatography. The resulting active fractions were analyzed by sodium dodecyl sulfate ("SDS")-polyacrylamide gel electrophoresis ("PAGE") and were found to have a molecular weight in the range of 12,000 to 16,000 daltons. Through isoelectrofocusing ("IEF") in polyacrylamide gels, the pI of the IL-1 was found to be in the range of from 5.0 to 5.4.
In a subsequent communication Mizel et al., 126 J. Immunol. 834-837 (1981), discussed purifying IL-1 from the same P388D.sub.1 cell line as used in Mizel, supra, to "apparent homogeneity" by ammonium sulfate precipitation, phenyl Sepharose chromatography, Ultrogel AcA54 gel filtration chromatography and preparative flat-bed IEF. From the IEF, the pI of the IL-1 was measured to be about 4.9 to 5.1. Through gel electrophoresis the molecular weight of the IL-1 molecule was determined to be approximately 14,000 daltons.
Researchers have also investigated IL-1 produced from human peripheral blood leukocytes and monocytes. Blyden et al., 118 J. Immunol. 1631-1638 (1977), disclosed a protocol for concentrating IL-1 prepared from human peripheral blood leukocytes by Sephadex G-100 column chromatography. This procedure was reported to result in a four-to-five fold concentration of the crude IL-1. DEAE-Bio-Gel A anion exchange chromatography was employed to remove the albumin from the serum used during the preparation of the crude IL-1. Next, the collected active fractions were adsorbed onto a hydroxylapatite column. Fractions containing peak IL-1 activity were then applied to a CM-Bio-Gel A cationic exchange resin. By these procedures, about 20% of the initial IL-1 was recovered. The resulting IL-1 was found to have a molecular weight of about 13,000 daltons and a pI of approximately 6.8 to 7.2.
Crude IL-1 prepared from human leukocytes by Togawa et al., 122 J. Immunol. 2112-2118 (1979) was initially processed by membrane filtration and then applied to a Bio-Gel P-100 chromatography column which disclosed two major peaks of activity, one in the range of from 12,000 to 22,000 daltons and another in the range of bout 50,000 to 70,000 daltons. Active fractions in the lower molecular weight region of the Bio-Gel P-100 column were pooled, applied to a Blue Sepharose column, and then applied to a DEAE-cellulose ion-exchange chromatography column. Thereafter, the IL-1 containing fractions were pooled and applied to a hydroxylapatite chromatography column. Togawa et al. discovered that when the lower molecular weight IL-1 activity resulting from each of these procedures was reconstituted with 2% human serum, concentrated and rechromatographed on Bio-Gel P-100, a significant portion of the higher molecular wight activity appeared.
In a more recent study, Lachman, 42 Federation Proceedings 2639-2645 (1983), reported preparing IL-1 by culturing peripheral blood monocytes or leukemic cells obtained from acute monocytic leukemia or acute myelomonocytic leukemia patients in a serum supplemented culture medium together with lipopolysaccharide ("LPS") to stimulate IL-1 production. Hollow fiber diafiltration and ultrafiltration were used to separate a lower molecular weight activity from most of the serum proteins. This lower weight activity was subjected to IEF in an Ampholine and sucrose gradient. From this procedure, the IL-1 activity was found to have a pI of about 6.8 to 7.2. The isoelectrofocused IL-1 activity was then subjected to SDS-PAGE which indicated that the human IL-1 being analyzed had a molecular weight of about 11,000 daltons. Lachman reported that the overall recovery of IL-1 activity from the above procedures was poor, in the range of about 4%.
Applicants have purified IL-1 to homogeneity using a combination of ion-exchange chromatographic procedures together with dye-ligand binding chromatography. Use of these procedures resulted in elaboration of a 17,500 dalton protein containing IL-1 activity. By trypsin degradation of the purified IL-1 protein, IL-1 peptides were liberated, one of which was subjected to amino acid sequence determination. Although applicants have been successful in purifying IL-1, the techniques developed for such purification remain costly and time consuming.
The availability of adequate quantities of homogeneous human IL-1 could be valuable in investigations and possible treatment of autoimmune disorders such as arthritis and lupus erythematosis. Also, human IL-1 in greater purity and larger quantities than heretofore available, could prove useful in achieving successful wound and burn healing.
One potential method of providing relatively large quantities of homogeneous human IL-1 is through recombinant DNA techniques. Recombinant DNA techniques have been developed for economically producing a desired protein once the gene coding for the protein has been isolated and identified. A discussion of such recombinant DNA techniques for protein production is set forth in the editorial and supporting papers in Vol. 196 of Science (April, 1977). However, to take advantage of the recombinant DNA techniques discussed in this reference, the gene coding for human IL-1 must first be isolated.